Gentamicin B (GB), a valuable starting material for the preparation of the semisynthetic aminoglycoside antibiotic isepamicin, is produced in trace amounts by the wild-type Micromonospora echinospora . While the biosynthetic pathway to GB has remained obscure for decades, we have now identified three hidden pathways to GB production via seven hitherto unknown intermediates in M. echinospora . The narrow substrate specificity of a key glycosyltransferase and the C6′-amination enzymes, in combination with the weak and unsynchronized gene expression of the 2′-deamination enzymes, limit GB production in M. echinospora . The crystal structure of the aminotransferase involved in C6′-amination explains its substrate specificity. Some of the new intermediates displayed similar premature termination codon readthrough activity but with reduced toxicity compared to the natural aminoglycoside G418. This work not only led to the discovery of unknown biosynthetic routes to GB, but also demonstrated the potential to mine new aminoglycosides from nature for drug discovery.

2-Deoxy-scyllo-inosose (DOI) has been a valuable starting natural product for the manufacture of pharmaceuticals or chemical engineering resources such as pyranose catechol. DOI synthase, which uses glucose-6-phosphate (Glc6P) as a substrate for DOI biosynthesis, is indispensably involved in the initial stage of the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics including butirosin, gentamicin, kanamycin, and tobramycin. A number of metabolically engineered recombinant strains of Bacillus subtilis were constructed here; either one or both genes pgi and pgcA that encode Glc6p isomerase and phosphoglucomutase, respectively, was (or were) disrupted in the sugar metabolic pathway of the host. After that, three different DOI synthase–encoding genes, which were artificially synthesized according to the codon preference of the B. subtilis host, were separately introduced into the engineered recombinants. The expression of a natural btrC gene, encoding DOI synthase in butirosin-producing B. circulans, in the heterologous host B. subtilis (BSDOI-2) generated approximately 2.3 g/L DOI, whereas expression of an artificially codon-optimized tobC gene, derived from tobramycin-producing Streptomyces tenebrarius, into the recombinant of B. subtilis (BSDOI-15) in which both genes pgi and pgcA are disrupted significantly enhanced the DOI titer: up to 37.2 g/L. Fed-batch fermentation by the BSDOI-15 recombinant using glycerol and glucose as a dual carbon source yielded the highest DOI titer (38.0 g/L). The development of engineered microbial cell factories empowered through convergence of metabolic engineering and synthetic biology should enable mass production of DOI. Thus, strain BSDOI-15 will surely be a useful contributor to the industrial manufacturing of various kinds of DOI-based pharmaceuticals and fine chemicals.

Aminoglycosides (AGs) are the first and foremost classical antibiotics that are biosynthesized by attachment of amino-sugar units via glycosidic bonds to aminocyclitol derivatives [1]. They are recognized to inhibit prokaryotic protein synthesis by binding to the 16S rRNA subunit of the 30S bacterial ribosome, thus causing irreversible bactericidal effects against the pathogens [2]. According to the chemical features of the aminocyclitol moiety and the core aglycone unit, AGs have been categorized into well-known types containing 2-deoxystreptamine (DOS), (including 4,5-disubstituted butirosins and neomycins or 4,6-disubstituted gentamicins and kanamycins) and 1,4diaminocyclitol-containing AGs [3]. In the late 1970s, the above 1,4-diaminocyclitol-containing AGs, namely fortimicins (FORs) and istamycins (ISTs), were isolated from Micromonospora olivasterospora and Streptomyces tenjimariensis, respectively [4,5]. The two share an unusual pseudodisaccharide moiety that is different from a common

Aminoglycosides (AGs) are broad‐spectrum antibiotics used to treat bacterial infections. Over the last two decades, studies have reported the potential of AGs in the treatment of genetic disorders caused by nonsense mutations, owing to their ability to induce the ribosomes to read through these mutations and produce a full‐length protein. However, the principal limitation in the clinical application of AGs arises from their high toxicity, including nephrotoxicity and ototoxicity. In this study, five novel pseudo‐trisaccharide analogs were synthesized by chemo‐enzymatic synthesis by acid hydrolysis of commercially available AGs, followed by an enzymatic reaction using recombinant substrate‐flexible KanM2 glycosyltransferase. The relationships between their structures and biological activities, including the antibacterial, nephrotoxic, and nonsense readthrough inducer (NRI) activities, were investigated. The absence of 1‐N‐acylation, 3′,4′‐dideoxygenation, and post‐glycosyl transfer modifications on the third sugar moiety of AGs diminishes their antibacterial activities. The 3′,4′‐dihydroxy and 6′‐hydroxy moieties regulate the in vitro nephrotoxicity of AGs in mammalian cell lines. The 3′,4′‐dihydroxy and 6′‐methyl scaffolds are indispensable for the ex vivo NRI activity of AGs. Based on the alleviated in vitro antibacterial properties and nephrotoxicity, and the highest ex vivo NRI activity among the five compounds, a kanamycin analog (6′‐methyl‐3′′‐deamino‐3′′‐hydroxykanamycin C) was selected as a novel AG hit for further studies on human genetic disorders caused by premature transcriptional termination.

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